Vaccine for the prevention of breast cancer recurrence

ABSTRACT

Provided are methods to induce and maintain a protective cytotoxic T-lymphocyte response to a peptide of the HER2/neu oncogene, GP2, with the effect of inducing and maintaining protective or therapeutic immunity against breast cancer in a patient in clinical remission, including patients having low to intermediate levels of HER2/neu expression. The methods comprise administering to the patient an effective amount of a vaccine composition comprising a pharmaceutically acceptable carrier, an adjuvant such as GM-CSF, and the GP2 peptide. The methods may further comprise administering a periodic booster vaccine dose as needed due to declining GP2-specific T cell immunity. Also provided are vaccine compositions for use in the methods.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on 7 Dec. 2009, is namedHMJ106PCT.txt, and is 11,473 bytes in size.

GOVERNMENT INTEREST

This invention was made in part with Government support. The Governmentmay have certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/133,487 (now U.S. Pat. No. 9,114,099), filed 8 Jun. 2011, which isthe U.S. National Phase application of International ApplicationPCT/US2009/067264, filed 9 Dec. 2009, which claims the benefit of, andrelies on the filing date of, U.S. provisional patent application No.61/121,220, filed 10 Dec. 2008, the entire disclosure of which isincorporated herein by reference.

BACKGROUND

Breast cancer (BCa) is the most common cancer diagnosis in women and thesecond-leading cause of cancer-related death among women (Ries L A G, etal. (eds). SEER Cancer Statistics Review, 1975-2003, National CancerInstitute, Bethesda, Md.). Major advances in breast cancer treatmentover the last 20 years have led to significant improvement in the rateof disease-free survival (DFS). For example, therapies utilizingantibodies reactive against tumor-related antigens have been used toblock specific cellular processes in order to slow disease progress orprevent disease recurrence. Despite the recent advances in breast cancertreatment, a significant number of patients will ultimately die fromrecurrent disease.

Vaccines are an attractive model for preventing, slowing, or prohibitingthe development of recurrent disease due to their ease ofadministration, and because of their high rate of success observed forinfectious diseases. The basic concept of constructing a cancer vaccineis straightforward in theory. The development of effective cancervaccines for solid tumors in practice, however, has met with limitedsuccess. For example, one group attempting to administer a peptidevaccine directed against metastatic melanoma observed an objectiveresponse rate of only 2.6% (Rosenberg S A et al. (2004) Nat. Med.10:909-15).

There are many potential explanations for this low success rate (CampoliM et al. (2005) Cancer Treat. Res. 123:61-88). For example, even if anantigen is specifically associated with a particular type of tumor cell,the tumor cells may express only low levels of the antigen, or it may belocated in a cryptic site or otherwise shielded from immune detection.In addition, tumors often change their antigenic profile by sheddingantigens as they develop. Also contributing to the low success rate isthe fact that tumor cells may express very low levels of MHC proteinsand other co-stimulatory proteins necessary to generate an immuneresponse.

Additional problems facing attempts at vaccination against tumors arisein patients with advanced-stage cancers. Such patients tend to havelarger primary and metastatic tumors, and the cells on the interior ofthe tumor may not be accessible due to poor blood flow. This isconsistent with the observation that vaccine strategies have tended tobe more successful for the treatment of hematologic malignancies(Radford K J et al. (2005) Pathology 37:534-50; and, Molldrem J J (2006)Biol. Bone Marrow Transplant. 12:13-8). In addition, as tumors becomemetastatic, they may develop the ability to release immunosuppressivefactors into their microenvironment (Campoli, 2005; and, Kortylewski Met al. (2005) Nature Med. 11:1314-21). Metastatic tumors have also beenassociated with a decrease in the number of peripheral bloodlymphocytes, and dendritic cell dysfunction (Gillanders W E et al.(2006) Breast Diseases: A Year Book and Quarterly 17:26-8).

While some or all of these factors may contribute to the difficulty indeveloping an effective preventative or therapeutic vaccine, the majorunderlying challenge is that most tumor antigens are self antigens orhave a high degree of homology with self antigens, and are thus expectedto be subject to stringent immune tolerance. Thus, it is clear that manypeptide-based cancer vaccines, with or without immune-stimulatingadjuncts, may be doomed to only limited success in clinical practice dueto low immunogenicity and lack of specificity.

Prototype breast cancer vaccines based on single antigens have beenmoderately successful in inducing a measurable immune response in animalexperiments and in clinical tests with breast cancer patients. Theobserved immune response, however, has not translated into aclinically-significant protective immunity against recurrence of diseaseput in remission by standard therapy (e.g., surgery, radiation therapy,and chemotherapy).

HER2/neu is a proto-oncogene expressed in many epithelial malignancies(Slamon D J et al. (1989) Science 244:707-12). HER2/neu is a member ofthe epidermal growth factor receptor family and encodes a 185-kdtyrosine kinase receptor involved in regulating cell growth andproliferation. (Popescu N C et al. (1989) Genomics 4:362-366; Yarden Yet al. (2001) Nat Rev Mol Cell Bio 2:127-137.) Over-expression and/oramplification of HER2/neu is found in 25-30% of invasive breast cancers(BCa) and is associated with more aggressive tumors and a poorerclinical outcome. (Slamon D J et al. Science (1987) 235:177-182; SlamonD J et al. Science (1989) 244:707-12; Toikkanen S et al. J Clin Oncol(1992) 10:1044-1048; Pritchard K I et al. (2006) N. Engl. J. Med.354:2103-11.)

Determining HER2/neu status is performed predominately via two tests,immunohistochemistry (IHC) and fluorescence in situ hybridization(FISH). IHC detects over-expression of HER2/neu protein and is reportedon a semi-quantitative scale of 0 to 3+(0=negative, 1⁺=low expression,2⁺=intermediate, and 3⁺=over-expression). FISH on the other hand detectsamplification (excess copies) of the HER2/neu gene and is expressed as aratio of HER2/neu gene copies to chromosome 17 gene copies andinterpreted as “over-expression” if FISH is ≧2.0 copies. (Hicks D G etal. Hum Pathol (2005) 36:250-261.) Concurrence rate of IHC and FISH isapproximately 90%. (Jacobs et al. J Clin Oncol (1999) 17:1533-1541.)FISH is considered the gold standard, as retrospective analysis revealsit is a better predictor of trastuzumab (Tz) response; it is moreobjective and reproducible. (Press M F et al. J Clin Oncol (2002)14:3095-3105; Bartlett J et al. J Pathol (2003) 199:411-417; Wolff A Cet al. J Clin Oncol (2007) 25:118-145.)

Identification and quantification of HER2/neu as a proto-oncogene hasled to humoral or antibody-based passive immunotherapy, including theuse of trastuzumab (Herceptin® Genentech Inc., South San Francisco,Calif.). Trastuzumab is a recombinant, humanized monoclonal antibodythat binds the extracellular juxtamembrane domain of HER2/neu protein.(Plosker G L et al. Drugs (2006) 66:449-475.) Tz is indicated forHER2/neu over-expressing (IHC 3⁺ or FISH≧2.0) node-positive (NP) andmetastatic BCa patients, (Vogel C L et al. J Clin Oncol (2002)20:719-726; Piccart-Gebhart M J et al. N Engl J Med (2005)353:1659-1672) and shows very limited activity in patients with low tointermediate HER2/neu expression. (Herceptin® (Trastuzumab),prescription product insert, Genentech Inc, South San Francisco, Calif.:revised September 2000.)

Another form of immunotherapy being pursued is vaccination and activeimmunotherapy targeting a cellular immune response to epitopes on tumorassociated antigens, such as HER2/neu. HER2/neu is a source of severalimmunogenic peptides that can stimulate the immune system to recognizeand kill HER2/neu-expressing cancer cells. (Fisk B et al. J Exp Med(1995) 181:2109-2117.) Two such peptides are termed E75 and GP2. E75 andGP2 are both nine amino-acid peptides that are human leukocyte antigen(HLA)-A2-restricted and stimulate CTL to recognize and lyseHER2/neu-expressing cancer cells (Fisk B et al. J Exp Med (1995)181:2109-2117; Peoples G E et al. Proc Natl Acad Sci USA (1995)92:432-436).

E75 is derived from the extracellular domain of the HER2/neu protein andcorresponds to amino acids 369-377 (KIFGSLAFL)(SEQ ID NO:3) of theHER2/neu amino acid sequence and is disclosed as SEQ ID NO:11 in U.S.Pat. No. 6,514,942, which patent is hereby incorporated by reference inits entirety. The full length HER2/neu protein sequence is set forthbelow and is disclosed as SEQ ID NO:2 in U.S. Pat. No. 5,869,445, whichpatent is hereby incorporated by reference in its entirety:

(SEQ ID NO: 1) MKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

Attempts have been made to utilize E75 as an anti-cancer vaccine, forexample, as a single peptide vaccine combined with differentimmunoadjuvants in patients with advanced cancer who overexpress theHER2/neu protein (Zaks T Z et al. (1998) Cancer Res. 58:4902-8; KnutsonK L et al. (2002) Clin. Cancer Res. 8:1014-8; and, Murray J L et al.(2002) Clin. Cancer Res. 8:3407-18); loaded on to autologous dendriticcells and reinfused (Brossart P et al. (2000) Blood 96:3102-8; and, KonoK et al. (2002) Clin. Cancer Res. 8:3394-3400); or embedded in longerpeptides capable of binding HLA class II molecules in order to recruitCD4 helper T-cells (Disis M L et al. (1999) Clin. Cancer Res. 5:1289-97;and, Disis M L et al. (2002) J. Clin. Oncol. 20:2624-32). Each approachhas stimulated an E75-specific cytotoxic T cell-mediated immuneresponse, but none has demonstrated a clinically significant therapeuticor protective immunity in women with advanced stage breast cancer. Theinability of others to show a meaningful clinical benefit using E75vaccine preparations stems in part from the fact that E75 is derivedfrom a “self” tumor antigen. Cancer vaccines targeting “self” tumorantigens, like those derived from HER2/neu, present unique challengesbecause of the immunologic tolerance characteristic of self proteins.Furthermore, the previous studies have focused on cancer patients withadvanced disease, such as Stage III or IV cancer, rather than patientswho are disease-free following standard therapies. As such, none ofthese attempts to use E75 as an anti-cancer vaccine has demonstrated thevaccine's ability to prevent or delay recurrence of disease followingremission. Building on these E75 studies, others have more recentlyconducted clinical trials to determine if E75-induced immunity conveys aclinical benefit by preventing the recurrence in high-risk breast cancerpatients. Peoples G E et al., J. Clin. Oncol. (2005) 23:7536-45; PeoplesG E et al., Clin Cancer Res (2008) 14(3):797-803; Holmes et al., Cancer(2008) 113:1666-75. The data from these studies indicate that increasedin vivo E75-induced DTH responses correlate with reduced recurrence andincreased survival time for those who did recur.

GP2, initially described by Peoples et al., is a nine amino acid peptidederived from the transmembrane portion of the HER2/neu proteincorresponding to amino acids 654-662 of the full length sequence (i.e.,IISAVVGIL: SEQ ID NO:2) (Peoples G E et al., Proc Natl Acad Sci USA(1995) 92:432-436, which is hereby incorporated by reference in itsentirety). The peptide was isolated using tumor-associated lymphocytesfrom patients with breast and ovarian cancer, and later found to beshared amongst several epithelial malignancies including non-small celllung cancer and pancreatic cancer (Linehan D C et al., J Immunol (1995)155:4486-4491; Peiper M et al., Surgery (1997) 122:235-242; Yoshino I etal., Cancer Res (1994) 54:3387-3390; Peiper M et al., Eur J Immunol(1997) 27:1115-1123).

E75 has a high binding affinity for the HLA-A2 molecule and isconsidered the immunodominant peptide of the HER2/neu protein. As suchit is the most studied HER2/neu-derived peptide in laboratory andclinical studies. Peoples et al., J. Clin. Oncol. (2005) 23:7536-45. Asthe immunodominant peptide, E75 is also expected to induce a more potentimmune response. GP2, on the other hand, has a relatively poor bindingaffinity to HLA-A2 and is considered a subdominant epitope. (Fisk B, etal. J Exp Med (1995) 181:2109-2117.) This is one of the reasons thatvaccine strategies targeting a cellular immune response to HER-2/neuepitopes have focused on E75 rather than GP2.

Previous studies of GP2 have used autologous dendritic cells pulsed withGP2 (and other peptides) ex vivo and re-injected subcutaneously(Brossart P et al. Blood (2000) 96:3102-3108) or intravenously (Dees E Cet al. Cancer Immunol Immunother (2004) 53:777-785) into HER2/neu⁺patients with metastatic breast or ovarian cancer to induce a CTLresponse. Brossart et al. detected a peptide-specific (GP2 and E75) CTLresponse in vivo, and they noted that both peptides showed a similarimmune response despite known differences in HLA-A2 binding affinities.Dees et al. evaluated GP2-pulsed dendritic cells in metastatic breastcancer patients and were able to document clinically stable disease intwo patients. Importantly, however, neither study used GP2 as a peptidevaccine. Rather in both studies, patients were injected with dendriticcells that had been pulsed with GP2. Furthermore, as with the E75studies, the GP2 studies were limited to patients with advanced cancer.Therefore, neither Brossart nor Dees demonstrated the ability ofGP2-pulsed dendritic cells to prevent or delay recurrence of diseasefollowing remission. As with E75, cancer vaccines targeting “self” tumorantigens, like HER2/neu from which GP2 is derived, present uniquechallenges because of the immunologic tolerance characteristic of selfproteins.

Peoples et al. have previously evaluated the use of GP2 for apeptide-based breast cancer vaccine trial by conducting in vitrocytotoxicity assays with GP2-pulsed dendritic cells and CD8 T cellsobtained from breast cancer patients. (Mittendorf E A et al. Cancer(2006) 106:2309-2317.) While the results from these in vitro experimentsconfirmed the presence of GP2-specific precursor cytotoxic T lymphocytesin women with HER2/neu+ breast cancer, it was concluded that because ofthe variability of response to a given peptide and the heterogeneity ofantigen expression in vivo, vaccination with multiple differentpeptides, including the immunodominant peptide E75, will be required toprovide an adequate immune response. (Mittendorf E A et al. Cancer(2006) 106:2309-2317.)

As noted above, trastuzumab is indicated for HER2/neu over-expressing(IHC 3⁺ or FISH≧2.0), node-positive (NP), metastatic breast cancerpatients, and shows very limited activity in patients with low tointermediate HER2/neu expression. Similarly, in the studies discussedabove, patients receiving the E75 and GP2-based vaccines were selected,in part, based on the presence of tumors that over-expressed HER2/neu.Accordingly, a GP2 peptide vaccine would not be expected to be effectivein cancer patients with low and intermediate levels of HER2/neu tumorexpression.

SUMMARY

In one embodiment, the invention features methods of preventing cancerrecurrence in a subject who has HER2/neu expressing tumor cells. In apreferred embodiment, the method is directed to preventing breast cancerrecurrence in a subject who is in remission following treatment with astandard course of therapy. In one embodiment, the standard course oftherapy is treatment with trastuzumab, which treatment may continueconcurrently with the methods described herein. The methods compriseadministering to the subject an effective amount of a compositioncomprising a pharmaceutically effective carrier and a GP2 peptide.Preferably the GP2 peptide has the amino acid sequence of SEQ ID NO:2.In one embodiment, other than the GP2 peptide, the composition does notcontain any other HER2/neu-derived peptides, including, for example, theimmunodominant peptide E75. The administration can be accomplished byany means suitable in the art, such as inoculation or injection, andmore particularly intradermal injection, which can occur with one ormore separate doses. Such doses may comprise an equal concentration ofthe peptide and an immunoadjuvant, may be administered substantiallyconcurrently, and can be administered at one inoculation site or spacedapart from each other on the surface of the skin. The composition can beadministered approximately three to six times or more on a monthly basisuntil a protective immunity is established. In some aspects, thecomposition further comprises an adjuvant such as granulocytemacrophage-colony stimulating factor (GM-CSF) and preferably recombinanthuman GM-CSF.

In some aspects, the methods further comprise administering to thesubject a booster vaccine dose, which comprises an effective amount of acomposition comprising a pharmaceutically effective carrier and apeptide having the amino acid sequence of SEQ ID NO:2. In some aspects,the composition of the booster further comprises an adjuvant such asGM-CSF and preferably recombinant human GM-CSF. The administration of abooster can be accomplished by any means suitable in the art, such asinoculation or injection, and more particularly intradermal injection,which can occur with one or more separate doses. Such doses may comprisean equal concentration of the peptide and an immunoadjuvant, may beadministered substantially concurrently, and can be administered at oneinoculation site or spaced apart from each other on the surface of theskin. Typically the booster is administered after a primary immunizationschedule has been completed, and preferably every six or 12 months afterthe primary immunization, as needed.

The subject can be any mammal, and is preferably a human. In certainaspects, the human is positive for major histocompatibility antigenblood-typed as human leukocyte antigen A2 or human leukocyte antigen A3.In other aspects, cancer cells from the human are positive for theexpression of detectable levels of HER2/neu. In some aspects, the cancercells exhibit low or intermediate expression of HER2/neu. For example,in some preferred aspects, the cancer cells from the human have animmunohistochemistry (IHC) rating of 1+ or 2+ and/or a fluorescence insitu hybridization (FISH) rating of less than 2.0). In other aspects,the cancer cells from the human may have an IHC rating up to 3+. Inother aspects, the cancer cells from the human can exhibitover-expression of HER2/neu. For example, in some preferred aspects, thecancer cells from the human have an immunohistochemistry (IHC) rating of3+ and/or a fluorescence in situ hybridization (FISH) rating of greaterthan or equal to 2.0). In other embodiments, the human does not havepre-existing immunity to GP2 (SEQ ID NO:2 or SEQ ID NO:4).

In another embodiment, the invention provides compositions for use inthe methods described in this application. In one aspect, thecompositions comprise a pharmaceutically acceptable carrier, aneffective amount of a peptide having the amino acid sequence of SEQ IDNO:2, and an adjuvant, such as granulocyte macrophage-colony stimulatingfactor. The compositions are preferably administered in an optimizedimmunization schedule. In one embodiment, the vaccine compositioncomprises 0.1-1 mg/ml peptide and 0.125-0.5 mg/ml adjuvant. In somespecific aspects, the preferred concentrations and schedules of thevaccine composition include: (1) 1 mg/ml peptide and 0.25 mg/mladjuvant, (2) 0.5 mg/ml peptide and 0.25 mg/ml adjuvant, (3) 0.1 mg/mlpeptide and 0.25 mg/ml adjuvant, (4) 1 mg/ml peptide and 0.125 mg/mladjuvant, and (5) 0.5 mg/ml peptide and 0.125 mg/ml adjuvant, each withmonthly inoculations for at least 6 consecutive months followed byperiodic booster inoculations (preferably semi-annually or annually) for1 year, 2 years, or 3 or more years.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate aspects of the invention andtogether with the description serve to explain the principles of theinvention. In the drawings:

FIGS. 1A-D show the mean local reactions vs. GM-CSF dose by dose groups.Patients were vaccinated with peptide and GM-CSF in four dose groups.FIG. 1A. 100 mcg peptide/250 mcg GM-CSF dose group. FIG. 1B. 500 mcgpeptide/250 mcg GM-CSF dose group. FIG. 1C. 1000 mcg peptide/250 mcgGM-CSF dose group. FIG. 1D. 500 mcg peptide/125 mcg GM-CSF dose group.Local reactions were measured in millimeters (solid lines). A localreaction ≧100 mm induration necessitated a 50% dose reduction in GM-CSFdose (dashed lines). There were no peptide dose reductions.

FIGS. 2A-C show toxicity and immunologic responses of all patientsenrolled in GP2 phase I trial. FIG. 2A. Toxicity—no patients experiencegrade 3-5 local or systemic toxicities. FIG. 2B. Ex vivo immuneresponse—pre-max % specific CD8⁺ T-cells statistically increased(p=0.001). FIG. 2C. In vivo immune response—GP2 pre-post DTHstatistically increased (p=0.0002). Normal saline (NS) control alsoshown for comparison.

FIGS. 3A-C show toxicity and immunologic responses of patients enrolledin GP2 phase I trial comparing no pre-existing immunity (pre-dimer<0.03) vs. pre-existing immunity (pre dimer >0.03). FIG. 3A.Toxicity—toxicities were slightly increased in the no pre-existingimmunity patients, albeit not significantly. FIG. 3B. Ex vivo immuneresponse—Patients without pre-existing immunity showed statisticallysignificant increases in pre-max, pre-post, and pre-long term % specificCD8⁺ T-cells (p=0.003, p=0.03, and p=0.01 respectively) in response tovaccination. FIG. 3C. In vivo immune response—both pre-post DTHresponses statistically increased (None p=0.03 and Pre-E p=0.0004). Nostatistical difference between post DTH responses was noted (p=0.3).

FIGS. 4A-C show toxicity and immunologic responses of patients enrolledin GP2 phase I trial comparing GM-CSF dose 125 mcg vs. 250 mcg. FIG. 4A.Toxicity—toxicities were slightly increased in the GM-CSF 250 mcgpatients, albeit not significantly. FIG. 4B. Ex vivo immune response—theGM-CSF 125 mcg pre-max % specific CD8⁺ T-cells did not statisticallyincrease (p=0.17), but the GM-CSF 250 mcg pre-max % specific CD8⁺T-cells did statistically increase (p=0.005). FIG. 4C. In vivo immuneresponse—GM-CSF 125 mcg and 250 mcg pre-post DTH statistically increased(respectively, p=0.009 and p=0.008). There was no statisticalsignificance between GM-CSF 125 mcg post DTH and GM-CSF 250 mcg post DTH(p=0.1).

FIG. 5 shows ex vivo immune response and epitope spreading in responseto GP2. Mean E75 specific CD8⁺ T-lymphocytes were measured in responseto vaccination with GP2 peptide vaccine. Pre-vaccination E75 dimer vs.maximum (0.8±0.2% vs. 2.0±0.2%, p=0.0001), pre vs. post (0.8±0.2% vs.1.2±0.2%, p=0.1), and pre vs. long-term (0.8±0.2% vs. 1.0±0.2%; p=0.6)were compared. No statistical differences between GP2 and E75 valueswere noted, but a trend towards larger E75 maximum dimer response wasnoted (2.0±0.2% vs. 1.4±0.2%; p=0.07).

FIG. 6 shows the in vivo immune response of patients enrolled in GP2phase II trial. Median DTH reaction to GP2 increased significantly frompre-vaccination level to post-vaccination level in the GP2 peptide group(PG) (1.0±0.8 cm to 18.0±3.1 cm; p<0.0001) and to a lesser extent in thecontrol, adjuvant group (AG) (0.0±1.0 cm to 0.5±3.3 cm; p<0.01). Thepost-vaccination DTH was significantly larger in the PG compared to theAG (18.0±3.1 cm vs 0.5±3.3 cm, p=0.002).

DETAILED DESCRIPTION

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

The term “prevent” or “prevention” refers to any success or indicia ofsuccess in the forestalling or delay of breast cancer recurrence/relapsein patients in clinical remission, as measured by any objective orsubjective parameter, including the results of a radiological orphysical examination.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound, material,or composition, as described herein effective to achieve a particularbiological result such as, but not limited to, biological resultsdisclosed, described, or exemplified herein. Such results may include,but are not limited to, the prevention of breast cancer, and moreparticularly, the prevention of recurrent breast cancer, e.g., theprevention of relapse in a subject, as determined by any means suitablein the art. Optimal therapeutic amount refers to the dose, schedule andthe use of boosters to achieve the best therapeutic outcome.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

“Protective immunity” or “protective immune response,” means that thesubject mounts an active immune response to an immunogenic component ofan antigen such as the breast cancer antigens described and exemplifiedherein, such that upon subsequent exposure to the antigen, the subject'simmune system is able to target and destroy cells expressing theantigen, thereby decreasing the incidence of morbidity and mortalityfrom recurrence of cancer in the subject. Protective immunity in thecontext of the present invention is preferably, but not exclusively,conferred by T lymphocytes.

“Pre-existing immunity” is defined as a peptide-specific dimer level ofat least 0.3%. The peptide-specific dimer level can be measured usingstandard assays, such as the HLA-A2 immunoglobulin dimer assay describedin this application.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Peptide” refers to any peptide comprising two or more amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres. Polypeptide refers to both short chains, commonlyreferred to as peptides, oligopeptides or oligomers, and to longerchains, generally referred to as proteins. Polypeptides may containamino acids other than the 20 gene-encoded amino acids. Polypeptidesinclude amino acid sequences modified either by natural processes, suchas post-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Polypeptides may be branched as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched and branched cyclic polypeptides may resultfrom natural posttranslational processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

“Booster” refers to a dose of an immunogen administered to a patient toenhance, prolong, or maintain protective immunity and to overcome thedown-regulation of T-cell responses mediated by regulatory T-cells.

“Free of breast cancer” or “disease free” or NED (No Evidence ofDisease) means that the patient is in clinical remission induced bytreatment with the current standard of care therapies. By “remission” or“clinical remission,” which are used synonymously, it is meant that theclinical signs, radiological signs, and symptoms of breast cancer havebeen significantly diminished or have disappeared entirely based onclinical diagnostics, although cancerous cells may still exist in thebody. Thus, it is contemplated that remission encompasses partial andcomplete remission. The presence of residual cancer cells can beenumerated by assays such as CTC (Circulating Tumor Cells) and may bepredictive of recurrence.

“Relapse” or “recurrence” or “resurgence” are used interchangeablyherein, and refer to the radiographic diagnosis of return, or signs andsymptoms of return of breast cancer after a period of improvement orremission.

Breast cancer is a major health concern for women worldwide. Breastcancer vaccines that have been attempted to date have been limited inefficacy, particularly with respect to preventing relapse in patientswho are in remission following a standard course of therapy. Asdiscussed in this application, it has been determined that administeringa peptide of the HER2/neu oncogene, GP2 (SEQ ID NO:2), can induce apotent in vivo immune response that is known to correlate with a reducedrate of recurrence of breast cancer in disease-free patients.

The GP2 peptide is associated with MHC HLA-A2, and thus may induceprotective immunity in patients having the HLA-A2 haplotype. The HLA-A2haplotype has been implicated as a negative prognostic factor in ovarian(Gamzatova et al., Gynecol Oncol (2006) 103:145-50) and prostate cancer(Hueman et al., Clin Cancer Res (2005) 11:7470-79; De Petris et al., MedOncol (2004) 21:49-52) and this finding likely extends to breast canceras well. Thus, HLA-A2⁺ patients seem to represent a higher risk todevelop cancer recurrence following remission. Nevertheless, it wasunexpectedly demonstrated that a vaccine composition comprisingGP2+GM-CSF effectively induced a potent in vivo immune response inHLA-A2⁺ patients that is known to correlate with a lower risk of breastcancer recurrence and longer disease-free survival as compared to HLA-ATcontrol patients. Moreover, it was surprisingly found that patientstreated with GP2 (the subdominant epitope) and GM-CSF exhibited morerobust DTH responses as compared to vaccine compositions comprising E75(the immunodominant epitope) and GM-CSF. Notably, these results were notobtained by combining GP2 with another epitope, such as E75 to generatea multiepitope vaccine, but instead were obtained with a single epitope(i.e., GP2) vaccine. In addition, based on preliminary data, it appearsthat GP2 can also induce protective immunity in patients having theHLA-A3 haplotype.

Because GP2 is derived from the HER2/neu protein, one would expect thatpatients overexpressing HER2/neu would exhibit a better response to aGP2-based vaccine than those with low to intermediate HER2/neuexpression. For example, another HER2/neu based therapy, trastuzumab(Herceptin® Genentech Inc., South San Francisco, Calif.), is onlyindicated for HER2/neu over-expressing (IHC 3⁺ or FISH≧2.0),node-positive (NP), metastatic breast cancer patients, and shows verylimited activity in patients with low to intermediate HER2/neuexpression. Nevertheless, it was unexpectedly observed that patientshaving low to intermediate levels of HER2/neu expression experiencedpotent immune responses to GP2, similar in magnitude to GP2-inducedresponses in patients overexpressing HER2/neu.

Accordingly, one embodiment of the present invention features vaccinecompositions for inducing protective immunity against breast cancerrelapse or recurrence. Another embodiment provides methods for inducingand for maintaining protective immunity against breast cancer, and moreparticularly against recurrent breast cancer. In some aspects, themethods comprise administering to a subject an effective amount of acomposition comprising a pharmaceutically effective carrier, apolypeptide having the amino acid sequence of SEQ ID NO:2, andoptionally an immunoadjuvant, such as GM-CSF. Variants of SEQ ID NO:2,including those with modified side chains of amino acids as described byU.S. Pat. Publ. No. 20050169934, which is hereby incorporated byreference in its entirety, are suitable for use in the vaccinecompositions and methods of this application.

In addition, a naturally occurring polymorphism at codon 655 (isoleucineto valine substitution) has been identified, yielding a polymorphic GP2peptide having the sequence IVSAVVGIL (SEQ ID NO:4) (Papewalis et al.,Nucleic Acid Res. (1991) 19:5452). This polymorphic GP2 peptide is alsosuitable for use in the vaccine compositions and methods of thisapplication. Similarly, several groups have investigated single, double,and triple amino-acid substitutions introduced at various sites in theGP2 peptide, including the anchor residues (positions 2 and 9), andfound that certain amino acid substitutions lead to increased binding ofGP2 to HLA-A2 (Tanaka et al., Int J Cancer (2001) 94:540-44; Kuhns etal., J Biol Chem (1999) 274:36422-427; Sharma et al., J Biol Chem (2001)276:21443-449, each of these references is hereby incorporated byreference in its entirety). Thus, one of skill in the art wouldunderstand that certain substitutions, particularly at the anchorresidues, could be made to GP2 without negatively affecting its abilityto induce a protective immune response. In one embodiment, the GP2peptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4except for a substitution at a residue that increases the affinity ofthe GP2 peptide for the HLA-A2 molecule. Preferably, the substitutionoccurs at one or both of the anchor residues of GP2 (positions 2 and 9).More preferably, the substitution comprises an isoleucine to leucinesubstitution at position 2 and/or a leucine to valine substitution atposition nine. In another embodiment, the GP2 peptide comprises theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 except for asubstitution at a residue that does not affect the affinity of the GP2peptide for the HLA-A2 molecule as compared to the affinity of a wildtype GP2 peptide comprising SEQ ID NO:2 for the HLA-A2 molecule. Assaysto test for binding affintity between GP2 and HLA-A2 are well-known inthe art and include, for example, the T2 cell surface assembly assaydisclosed in Sharma et al., J Biol Chem (2001) 276:21443-449.

In one aspect, the GP2 peptide has no more than 9, 10, 11, 12, 13, 14,or 15 amino acid residues. In one embodiment, the GP2 peptide has nomore than 9 amino acid residues. Preferably, the GP2 peptide with nomore than 9 amino acids is SEQ ID NO:2 or SEQ IDNO:4, or a mutantversion of SEQ ID NO:2 or SEQ ID NO:4 with a substitution at position 2and/or 9.

The subject can be any animal, and preferably is a mammal such as ahuman, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow,horse, pig, and the like. Humans are most preferred. In highly preferredaspects, the humans are positive for the HLA-A2 haplotype. In otherpreferred aspects, the humans are positive for the expression of humanHER2/neu, including preferentially humans with low and/or intermediateHER2/neu expressing tumors, as well as humans that are overexpressors ofHER2/neu.

Additionally, our group has previously demonstrated a possible synergybetween trastuzumab and GP2-peptide stimulated CTLs ex vivo.Pre-treatment of breast cancer cells with trastuzumab followed byincubation with GP2-peptide induced CTLs resulted in enhancedcytotoxicity in three tumor cell lines compared to treatment withtrastuzumab or GP2-specific CTLs alone (Mittendorf E A et al., Annals ofSurgical Oncology (2006) 13(8):1085-1098). In view of the results fromthe experiments with GP2 disclosed in this application, these findingsindicate that concurrent GP2 vaccination during trastuzumab therapy maybe an effective combination immunotherapy.

The vaccine compositions can be formulated as freeze-dried or liquidpreparations according to any means suitable in the art. Non-limitingexamples of liquid form preparations include solutions, suspensions,syrups, slurries, and emulsions. Suitable liquid carriers include anysuitable organic or inorganic solvent, for example, water, alcohol,saline solution, buffered saline solution, physiological salinesolution, dextrose solution, water propylene glycol solutions, and thelike, preferably in sterile form.

The vaccine compositions can be formulated in either neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the active polypeptides) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from free carboxyl groups can alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

The vaccine compositions are preferably formulated for inoculation orinjection into the subject. For injection, the vaccine compositions ofthe invention can be formulated in aqueous solutions such as water oralcohol, or in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, preserving,stabilizing and/or dispersing agents. Injection formulations may also beprepared as solid form preparations which are intended to be converted,shortly before use, to liquid form preparations suitable for injection,for example, by constitution with a suitable vehicle, such as sterilewater, saline solution, or alcohol, before use.

The vaccine compositions can also be formulated in sustained releasevehicles or depot preparations. Such long acting formulations may beadministered by inoculation or implantation (for example subcutaneouslyor intramuscularly) or by injection. Thus, for example, the vaccinecompositions may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well-known examplesof delivery vehicles suitable for use as carriers.

The vaccine compositions can comprise agents that enhance the protectiveefficacy of the vaccine, such as adjuvants. Adjuvants include anycompound or compounds that act to increase a protective immune responseto the GP2 peptide antigen, thereby reducing the quantity of antigennecessary in the vaccine, and/or the frequency of administrationnecessary to generate a protective immune response. Adjuvants caninclude for example, emulsifiers, muramyl dipeptides, avridine, aqueousadjuvants such as aluminum hydroxide, chitosan-based adjuvants, and anyof the various saponins, oils, and other substances known in the art,such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpGsequences, synthetic oligonucleotides and combinations thereof (Schijnset al. (2000) Curr. Opin. Immunol 12:456), Mycobacterialphlei (M. phlei)cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA(M-DNA), and M-DNA-M. phlei cell wall complex (MCC). Compounds which canserve as emulsifiers include natural and synthetic emulsifying agents,as well as anionic, cationic and nonionic compounds. Among the syntheticcompounds, anionic emulsifying agents include, for example, thepotassium, sodium and ammonium salts of lauric and oleic acid, thecalcium, magnesium and aluminum salts of fatty acids, and organicsulfonates such as sodium lauryl sulfate. Synthetic cationic agentsinclude, for example, cetyltrhethylammonlum bromide, while syntheticnonionic agents are exemplified by glycerylesters (e.g., glycerylmonostearate), polyoxyethylene glycol esters and ethers, and thesorbitan fatty acid esters (e.g., sorbitan monopalmitate) and theirpolyoxyethylene derivatives (e.g., polyoxyethylene sorbitanmonopalmitate). Natural emulsifying agents include acacia, gelatin,lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as asingle oil, a mixture of oils, a water-in-oil emulsion, or anoil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, oran animal oil. Mineral oils are liquid hydrocarbons obtained frompetrolatum via a distillation technique, and are also referred to in theart as liquid paraffin, liquid petrolatum, or white mineral oil.Suitable animal oils include, for example, cod liver oil, halibut oil,menhaden oil, orange roughy oil and shark liver oil, all of which areavailable commercially. Suitable vegetable oils, include, for example,canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil,safflower oil, sesame oil, soybean oil, and the like. Freund's CompleteAdjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) are two commonadjuvants that are commonly used in vaccine preparations, and are alsosuitable for use in the present invention. Both FCA and FIA arewater-in-mineral oil emulsions; however, FCA also contains a killedMycobacterium sp.

Immunomodulatory cytokines can also be used in the vaccine compositionsto enhance vaccine efficacy, for example, as an adjuvant. Non-limitingexamples of such cytokines include interferon alpha (IFN-α),interleukin-2 (IL-2), and granulocyte macrophage-colony stimulatingfactor (GM-CSF), or combinations thereof. GM-CSF is highly preferred.

Vaccine compositions comprising GP2 peptide antigens and furthercomprising adjuvants can be prepared using techniques well known tothose skilled in the art including, but not limited to, mixing,sonication and microfluidation. The adjuvant can comprise from about 10%to about 50% (v/v) of the vaccine composition, more preferably about 20%to about 40% (v/v), and more preferably about 20% to about 30% (v/v), orany integer within these ranges. About 25% (v/v) is highly preferred.

Administration of the vaccine compositions can be by infusion orinjection (e.g., intravenously, intramuscularly, intracutaneously,subcutaneously, intrathecal, intraduodenally, intraperitoneally, and thelike). The vaccine compositions can also be administered intranasally,vaginally, rectally, orally, or transdermally. Additionally, vaccinecompositions can be administered by “needle-free” delivery systems.Preferably, the compositions are administered by intradermal injection.Administration can be at the direction of a physician or physicianassistant.

The injections can be split into multiple injections, with such splitinoculations administered preferably substantially concurrently. Whenadministered as a split inoculation, the dose of the immunogen ispreferably, but not necessarily, proportioned equally in each separateinjection. If an adjuvant is present in the vaccine composition, thedose of the adjuvant is preferably, but not necessarily, proportionedequally in each separate injection. The separate injections for thesplit inoculation are preferably administered substantially proximal toeach other on the patient's body. In some preferred aspects, theinjections are administered at least about 1 cm apart from each other onthe body. In some preferred aspects, the injections are administered atleast about 2.5 cm apart from each other on the body. In highlypreferred aspects, the injections are administered at least about 5 cmapart from each other on the body. In some aspects, the injections areadministered at least about 10 cm apart from each other on the body. Insome aspects, the injections are administered more than 10 cm apart fromeach other on the body, for example, at least about 12.5. 15, 17.5, 20,or more cm apart from each other on the body. Primary immunizationinjections and booster injections can be administered as a splitinoculation as described and exemplified herein.

Various alternative pharmaceutical delivery systems may be employed.Non-limiting examples of such systems include liposomes and emulsions.Certain organic solvents such as dimethylsulfoxide also may be employed.Additionally, the vaccine compositions may be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic agent. The various sustained-releasematerials available are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the vaccine compositions over a range of several days to severalweeks to several months.

To prevent breast cancer recurrence in a patient who is in breast cancerremission, a therapeutically effective amount of the vaccine compositionis administered to the subject. A therapeutically effective amount willprovide a clinically significant increase in the number of GP2-specificcytotoxic T-lymphocytes (CD8⁺) in the patient, as well as a clinicallysignificant increase in the cytotoxic T-lymphocyte response to theantigen, as measured by any means suitable in the art. In addition, dueto epitope spreading, a therapeutically effective amount of the GP2vaccine composition will provide an increase in the number ofE75-specific cytotoxic T-lymphocytes (CD8⁺) in the patient, as measuredby any means suitable in the art. In the patient on the whole, atherapeutically effective amount of the vaccine composition will destroyresidual microscopic disease and significantly reduce or eliminate therisk of recurrence of breast cancer in the patient.

The effective amount of the vaccine composition may be dependent on anynumber of variables, including without limitation, the species, breed,size, height, weight, age, overall health of the patient, the type offormulation, the mode or manner or administration, or the presence orabsence of risk factors that significantly increase the likelihood thatthe breast cancer will recur in the patient. Such risk factors include,but are not limited to the type of surgery, status of lymph nodes andthe number positive, the size of the tumor, the histologic grade of thetumor, the presence/absence of hormone receptors (estrogen andprogesterone receptors), HER2/neu expression, lymphovascular invasion,and genetic predisposition (BRCA 1 and 2). In some preferred aspects,the effective amount is dependent on whether the patient is lymph nodepositive of lymph node negative, and if the patient is lymph nodepositive, the number and extent of the positive nodes. In all cases, theappropriate effective amount can be routinely determined by those ofskill in the art using routine optimization techniques and the skilledand informed judgment of the practitioner and other factors evident tothose skilled in the art. Preferably, a therapeutically effective doseof the vaccine compositions described herein will provide thetherapeutic preventive benefit without causing substantial toxicity tothe subject.

Toxicity and therapeutic efficacy of the vaccine compositions can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Vaccine compositions that exhibit large therapeutic indicesare preferred. Data obtained from cell culture assays and animal studiescan be used in formulating a range of dosage for use in patients. Thedosage of such vaccine compositions lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized.

Toxicity information can be used to more accurately determine usefuldoses in a specified subject such as a human. The treating physician canterminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions, and can adjust treatment as necessary if theclinical response is not adequate, to improve the response. Themagnitude of an administrated dose in the prevention of recurrent breastcancer will vary with the severity of the patient's condition, relativerisk for recurrence, or the route of administration, among otherfactors. The severity of the patient's condition may, for example, beevaluated, in part, by standard prognostic evaluation methods.

The vaccine compositions can be administered to a patient on anyschedule appropriate to induce and/or sustain protective immunityagainst breast cancer relapse, and more specifically to induce and/orsustain a cytotoxic T lymphocyte response to GP2 and/or E75 (due toepitope spreading). For example, patients can be administered a vaccinecomposition as a primary immunization as described and exemplifiedherein, followed by administration of a booster to bolster and/ormaintain the protective immunity.

In some aspects, patients can be administered the vaccine compositions1, 2 or more times per month. Once per month for six consecutive monthsis preferred to establish the protective immune response, particularlywith respect to the primary immunization schedule. In some aspects,boosters can be administered at regular intervals such as every 6 ormore months after completion of the primary immunization schedule.Administration of the booster is preferably every 6 months. Boosters canalso be administered on an as-needed basis.

The vaccine administration schedule, including primary immunization andbooster administration, can continue as long as needed for the patient,for example, over the course of several years, to over the lifetime ofthe patient. In some aspects, the vaccine schedule includes morefrequent administration at the beginning of the vaccine regimen, andincludes less frequent administration (e.g., boosters) over time tomaintain the protective immunity.

The vaccine can be administered at lower doses at the beginning of thevaccine regimen, with higher doses administered over time. The vaccinescan also be administered at higher doses at the beginning of the vaccineregimen, with lower doses administered over time. The frequency ofprimary vaccine and booster administration and dose of GP2 administeredcan be tailored and/or adjusted to meet the particular needs ofindividual patients, as determined by the administering physicianaccording to any means suitable in the art.

In some aspects, the vaccine compositions, including compositions foradministration as a booster, comprise from about 0.1 mg to about 10 mgof GP2 peptide. In some preferred aspects, the compositions compriseabout 0.1 mg of GP2. In some preferred aspects, the compositionscomprise about 1 mg of GP2. In some most preferred aspects, thecompositions comprise about 0.5 mg of GP2.

In some preferred aspects, the vaccine compositions comprising GP2,including compositions for administration as a booster, further compriseGM-CSF. Such compositions preferably comprise from about 0.01 mg toabout 0.5 mg of GM-CSF. In some preferred aspects, the compositionscomprise about 0.125 mg of GM-CSF. In some preferred aspects, thecompositions comprise about 0.25 mg of GM-CSF.

In some particularly preferred aspects, the vaccine compositionscomprise about 0.5 mg to 1 mg of GP2 peptide and from 0.125 to 0.250 mgof GM-CSF in a total volume of 1 ml, and are administered monthly as asplit inoculation of 0.5 ml each, administered by injections about 5 cmapart on the patient's body, and administered concurrently or admixed.The administration schedule is preferably monthly for six months. Aftera period of about 48 hours, the injection site can be assessed for localreaction of erythema and induration. If the reactions at both sites areconfluent and the area of total induration measures >100 mm (or thepatient experiences any >grade 2 systemic toxicity), then the dose ofGM-CSF may be reduced, for example, by half, though it is intended thatthe peptide dose remain the same. If the patient presents a robustreaction on subsequent doses, then further reduction of GM-CSF canoccur, for example, reducing by half. If the patient does not presentwith a robust reaction, then the patient can continue with the higherGM-CSF dose. In some aspects, the administration schedule and dosing ofthe booster is similarly determined, with boosters beginning withadministration of vaccine compositions comprising 1 mg of GP2 and 0.25mg GM-CSF, administered about every six months following the conclusionof the primary immunization vaccine schedule.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Phase I Trial of GP2+GM-CSF

Patient Characteristics and Clinical Protocol:

This is the first phase I clinical trial of the HER2/neu-derivedGP2-peptide with the GM-CSF immunoadjuvant in disease-free breast cancerpatients. The trial was Institutional Review Board-approved andconducted at Walter Reed Army Medical Center under an investigationalnew drug application (BB-IND #11730). All patients had histologicallyconfirmed node-negative breast cancer that expressed all levels ofHER2/neu by standard immunohistochemistry (IHC 1-3+). Patients hadcompleted a standard course of surgery, chemotherapy, and radiationtherapy (as required) prior to enrollment, and those patients onhormonal chemoprevention were continued on their specific regimen. Afterscreening for eligibility criteria and proper counseling and consenting,eligible HLA-A2+ patients were enrolled into the study. Beforevaccination, patients were skin tested with a panel of recall antigens(Mantoux test). Patients were considered immunocompetent if they reacted(>5 mm) to ≧2 antigens.

We enrolled and vaccinated 18 node-negative, disease-free breast cancerpatients with all levels of HER2/neu expression (IHC 1-3+). No patientswithdrew from this study or were lost to follow up. Patientdemographics, prognostic factors, and treatment profiles are presentedin Table 1.

TABLE 1 Patient demographics, prognostic factors, and treatmentprofilesfor Phase I Study. GP2 Patients (n = 18) Median age, years 47Range, years 32-68 Race White, # (%) 14 (77.8)  Black, # (%) 2 (11.1)Other, # (%) 2 (11.1) Tumor size T2-T4, # (%) 7 (38.9) Histologicalgrade Grade III, # (%) 7 (38.9) HER2/neu IHC 3⁺ or FISH⁺, # (%) 6 (33.3)Hormone receptor negative, # (%) 8 (44.4) No chemotherapy, # (%) 6(33.3) No XRT, # (%) 6 (33.3) Hormonal therapy, # (%) 9 (50.0)

Vaccination and Clinical Protocol

Vaccine.

The GP2-peptide (HER2/neu, 654-662) was commercially produced inaccordance with federal guidelines for good manufacturing practices(GMP) by NeoMPS Inc. (San Diego, Calif.). Peptide purity (>95%) wasverified by high-performance liquid chromatography and massspectrometry, and the amino acid content was determined by amino acidanalysis. Sterility, endotoxin (limulus amebocyte lysate test), andgeneral safety testing was carried out by the manufacturer. Lyophilizedpeptide was reconstituted in sterile saline at the followingconcentrations: 100 mcg/0.5 ml, 500 mcg/0.5 ml, and 1 mg/0.5 ml. TheGP2-peptide was mixed with GM-CSF (Berlex, Seattle, Wash.) at 250mcg/0.5 ml, and the 1.0 ml inoculation was split and given intradermallyat two sites 5 cm apart in the same extremity.

Vaccination Series.

The study was designed and conducted as a dose escalation safety trialto determine the safety, immunogenicity, and optimal best dose of theGP2-peptide in combination with the adjuvant GM-CSF. The optimal bestdose was defined as the minimum dose of the vaccine and adjuvant thatgives the best in vivo and ex vivo immunologic response.

Three patients were assigned to each of the first three dose groupsreceiving six monthly inoculations of GP2 and 250 mcg of GM-CSF. Dosegroups are listed as GP2-peptide(mcg):GM-CSF(mcg):# of inoculations, andinclude: 100:250:6, 500:250:6, and 1000:250:6. GM-CSF was reduced by 50%if patients developed a local reaction measuring >100 mm or >grade 2systemic toxicities. In the last group of patients, GM-CSF was reducedto 125 mcg so that these nine patients received 500:125:6.

This dose escalation trial utilized an increasing GP2-peptide dose (100mcg, 500 mcg, and 1000 mcg) with 250 mcg of GM-CSF and 6 monthlyinoculations for the first three dose groups (abbreviated:GP2-peptide(mcg):GM-CSF(mcg):# inoculations—100:250:6, 500:250:6, and1000:250:6). The GM-CSF was reduced by 50% if patients developed a localreaction measuring >100 mm or >grade 2 systemic toxicities. Eight of thefirst 9 patients (89%) required GM-CSF dose reductions due to robustlocal reactions. Due to the number of dose reductions required, thestarting dose of GM-CSF was reduced from 250 mcg to 125 mcg perinoculation for the fourth and final group of 9 patients (500:125:6).Only 2 of the 9 patients (22%) in the final dose group required afurther GM-CSF dose reduction. No peptide dose reductions were requiredfor the vaccination series. FIG. 1 depicts the mean local reactions vs.the mean GM-CSF dose for each dose group. Local reactions in the finaldose group fluctuated less throughout the vaccination series using aGM-CSF starting dose of 125 mcg per inoculation.

Toxicity.

Patients were observed one hour post-vaccination for immediatehypersensitivity and returned 48-72 hours later to have their injectionsites measured and questioned about toxicities. Toxicities were gradedby the NCI Common Terminology Criteria for Adverse Events, v3.0 (CTCAE).Progression from one dose group to the next occurred only in the absenceof dose limiting toxicities, defined as hypersensitivity reaction or twopatients within a dose group developing ≧grade 3 toxicity.

Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cultures.

Blood was drawn before each vaccination and at one (post-vaccine) andsix months (long-term) after vaccine series completion. 50 ml of bloodwas drawn and PBMCs were isolated. PBMCs were washed and re-suspended inculture medium and used as a source of lymphocytes.

HLA-A2 Immunoglobulin Dimer Assay.

The presence of GP2-specific CD8+ T cells in freshly isolated PBMCs frompatients was assessed directly ex vivo by the dimer assay at baseline,prior to each successive vaccination, and at 1, 6, and 12 monthsfollowing completion of the vaccination series (Woll M M et al., J ClinImmunol (2004) 24:449-461). Briefly, the HLA-A2:Immunoglobulin (Ig)dimer (PharMingen, San Diego, Calif.) was loaded with the GP2, E75, orcontrol peptide (E37, folate binding protein (25-33) RIAWARTEL) byincubating 1 mcg of dimer with an excess (5 mcg) of peptide and 0.5 mcgof β2-microglobulin (Sigma, St. Louis, Mo.) at 37° C. overnight thenstored at 4° C. until used. PBMCs were washed and re-suspended inPharMingen Stain Buffer (PharMingen) and added at 5×10⁵ cells/100μl/tube in 5 ml round-bottom polystyrene tubes (Becton Dickinson,Mountain View, Calif.) and stained with the loaded dimers andantibodies. In each patient the level of GP2-specific and E75-specificCD8+cells was determined in response to each successive vaccination, andaverage post-inoculation levels compared to pre-inoculation levels.

Delayed Type Hypersensitivity (DTH).

DTH reactions to the GP2-peptide were performed prior to, and following,the vaccination series. Intradermal injections, on the back or extremity(opposite side from vaccination), using 100 mcg of GP2 (without GM-CSF)in 0.5 mL saline were compared to an equal volume control inoculum ofsaline. DTH reactions were measured in two dimensions at 48-72 hoursusing the sensitive ballpoint pen method and reported as the orthogonalmean. Sokol J E, Measurement of delayed skin test responses. N Engl JMed (1975) 293:501-501.

Statistical Analysis.

P values for clinicopathological factors were calculated using Wilcoxon,Fisher's exact test or χ² as appropriate. P values for comparing pre andpost-vaccination DTH and dimer assays were calculated using Studentt-test, paired or unpaired, as appropriate. Differences were consideredsignificant when p<0.05.

Results

Compositions comprising GP2 and GM-CSF are both safe and highlyimmunogenic. The immune responses, both ex vivo and in vivo, appear tobe influenced by the presence or absence of GP2-specific immunity at theinitiation of the inoculation series and by the GM-CSF dose utilized. Inaddition, GP2 vaccination efficiently results in intra-antigenic epitopespreading.

Toxicity was limited to mild local reactions (which are desired andserve as a surrogate measure of immunogenicity) and mild systemicresponses, many of which are known side effects of GM-CSF. There were nodose limiting toxicities, and dose reductions in GM-CSF were sufficientto limit the local reactions encountered with serial inoculations to≦grade 2. Overall the vaccine combination was well tolerated.

As discussed in further detail below, the ex vivo immunogenicity of thevaccine was demonstrated, but primarily evident when performing subgroupanalysis of the patients without pre-existing immunity. Patients withoutpre-existing immunity, as previously defined as peptide specific dimerlevel <0.3%, achieved the greatest induction of a CTL response to GP2vaccination. This response was uniform without regard to the dose ofGP2-peptide. Patients with pre-existing immunity demonstrated a lesserCTL response which suggests either a level of tolerance to the peptidevaccinations, or a previously optimized endogenous immune response.

The in vivo immunogenicity of the GP2+GM-CSF vaccine was demonstrated byan increase in the DTH reaction in response to the GP2-peptide (withoutGM-CSF) before and after the vaccination series. This difference inresponse reached statistical significance cumulatively and within eachdose group. Of note, patients without pre-existing immunity trendedtoward larger DTH reactions. Also, patients receiving the 250 mcg GM-CSFdose trended toward a larger DTH response, but this finding wasconfounded by a larger percentage of patients with pre-existing immunityin the lower GM-CSF dose group. Therefore, it is unclear if thedifference seen in the 250 mcg GM-CSF patients is due to the adjuvantdose or lack of tolerance. Together, these DTH responses would indicatethat in vivo immunity is maintained and augmented in all groups inresponse to vaccination.

Dose Groups.

This dose escalation trial utilized an increasing GP2-peptide dose (100mcg, 500 mcg, and 1000 mcg) with 250 mcg of GM-CSF and 6 monthlyinoculations for the first three dose groups (abbreviated:GP2-peptide(mcg):GM-CSF(mcg):# inoculations—100:250:6, 500:250:6, and1000:250:6). The GM-CSF was reduced by 50% if patients developed a localreaction measuring ≧100 mm or ≧grade 2 systemic toxicities. Eight of thefirst 9 patients (89%) required GM-CSF dose reductions due to robustlocal reactions. Due to the number of dose reductions required, thestarting dose of GM-CSF was reduced from 250 mcg to 125 mcg perinoculation for the fourth and final group of 9 patients (500:125:6).Only 2 of the 9 patients (22%) in the final dose group required afurther GM-CSF dose reduction. No peptide dose reductions were requiredfor the vaccination series. FIG. 1 depicts the mean local reactions vs.the mean GM-CSF dose for each dose group. Local reactions in the finaldose group fluctuated less throughout the vaccination series using aGM-CSF starting dose of 125 mcg per inoculation.

Combined Dosing Group.

There were no grade 3-5 toxicities among the 18 patients receiving atotal of 108 doses of GP2+GM-CSF. Among all patients, maximum localtoxicities occurring during the entire series were grade 1 (38.9%) orgrade 2 (61.1%). Maximum systemic toxicities during the series weregrade 0 (5.6%), grade 1 (61.1%), and grade 2 (33.3%). The most commonlocal reactions included erythema and induration (100% of patients),pruritis (25%), and inflammation (23%). The most common systemicreactions were grade 1 fatigue (40%) and grade 1 arthralgia/myalgia(15%). Overall combined local and systemic toxicity rates are noted inFIG. 2 a.

The GP2+GM-CSF vaccine was capable of eliciting an immune response bothex vivo and in vivo. Ex vivo immune response was assessed via HLA-A2:Igdimer assay to detect the percentage of circulating GP2-specific CD8⁺ Tcells. GP2-specific CTLs are reported as the mean±standard errorpercentage of the total circulating CD8⁺ population. Time pointsanalyzed include pre-vaccine (pre=0.5±0.1%), one month after completionof all inoculations (post=0.6±0.1%), maximum value during series(max=1.4±0.2%), and 6 months after completion of all inoculations(long-term=0.9±0.2%). While a statistically significant increaseoccurred in patients when comparing pre vs. maximum vaccine level(p=0.0003), no significant increase was seen comparing pre vs. post orlong-term vaccine dimer levels (p=0.7 and p=0.2, respectively) (FIG. 2b).

The vaccine's in vivo effectiveness was analyzed via pre andpost-vaccine series DTH responses using GP2 (without GM-CSF) as well asa saline volume control. A statistically significant increase was notedin GP2 pre vs. post-vaccine DTH responses (2.5±1.4 mm vs. 35.1±7.0 mm,p=0.0002) (FIG. 2 c).

To better elucidate the immunologic response to the GP2 vaccine, twodifferent sub-set analyses were performed: response based on thepresence of pre-existing GP2-specific immunity and response based ondose of GM-CSF. These are provided below.

Pre-Existing Vs. No Pre-Existing Immunity.

As previously defined, pre-existing immunity is a peptide specific dimerlevel >0.3% (Peoples G E et al., J Clin Oncol (2005) 23:7536-7545). Tenpatients (56%) had dimer levels consistent with pre-existing immunity toGP2, and 8 patients (44%) had no pre-existing immunity. There was astatistical difference between the two groups pre-vaccine GP2-dimerlevels (0.8+0.1% vs. 0.06+0.02%, p=0.0007).

Patients without pre-existing immunity had slightly increased localreactions with slightly higher local toxicities compared to the groupwith pre-existing immunity; although, this was not statisticallysignificant (FIG. 3 a).

Ex vivo and in vivo immune responses were observed in both groups, butwere more robust in the group of patients without pre-existing immunity.GP2 dimer levels from the group without pre-existing immunity were prevs. max (0.06±0.02% vs. 1.4±0.4%; p=0.009), pre vs. post (0.06±0.02% vs.0.5±0.2%; p=0.07), and pre vs. long-term (0.06±0.02% vs. 0.9±0.4%;p=0.06). In the 10 patients with pre-existing immunity, the CTL responseto vaccination was pre vs. max (0.8±0.1% vs. 1.5±0.2%; p=0.02), pre vs.post (0.8±0.1% vs. 0.6±0.2%; p=0.2), and pre vs. long-term (0.8±0.1 vs.0.9±0.2; p=0.7) (FIG. 3 b).

When comparing the groups in vivo immune responses both groups hadstatistically significant increases in their pre vs. post DTH responses(no pre-existing immunity=3.3±2.1 mm vs. 43.9±14.6 mm; p=0.02; andpre-existing immunity=2.0±2.0 mm vs. 28.0±4.6 mm; p=0.0001).

Patients without pre-existing immunity had larger post DTH responsescompared to the post DTH response of the group with pre-existingimmunity, but this difference was not statistically significant(43.9+14.6 mm vs. 28.0+4.6 mm, respectively; p=0.3) (FIG. 3 c).

GM-CSF 250 mcg vs. 125 mcg. Analysis of the patients according to thetwo starting doses of GM-CSF was also performed. Both local and systemictoxicities were decreased in the final dose group of 125 mcg GM-CSF,albeit not statistically significant (FIG. 4 a).

CTL response to vaccination in the 250 mcg dose groups (n=9) were prevs. max (0.3±0.1% vs. 1.1±0.2%; p=0.004), pre vs. post (0.3±0.1% vs.0.5±0.2%; p=0.07), and pre vs. long-term (0.3±0.1% vs. 0.4±0.09%;p=0.2). The CTL response in the 125 mcg dose group (n=9) was pre vs. max(0.8±0.2% vs. 1.8±0.3%; p=0.04), pre vs. post (0.8±0.2% vs. 0.6±0.2%;p=0.5), and pre vs. long-term (0.8±0.2% vs. 1.4±0.3%; p=0.5) (FIG. 4 b).Both the 250 mcg and 125 mcg groups of GM-CSF had significant increasesin pre to maximum dimer response, and the 250 mcg group trended towardssignificance. This analysis may be confounded by the fact that 33% (3/9)of the patients from the 250 mcg group had pre-existing immunity, while77.8% (7/9) patients from the 125 mcg group had pre-existing immunity.

For in vivo immune responses, all patients, regardless of GM-CSF dose,had a statistically significant increase in DTH response comparing prevs. post-vaccine measurements (125 mcg=3.8±2.5 mm to 24.4±5.5 mm;p=0.009, and 250 mcg=1.3±1.3 mm to 45.7±12.2 mm; p=0.008). Patientsreceiving 250 mcg of GM-CSF had a trend toward larger post-vaccine DTHresponses, albeit not statistically significant (45.7±12.2 mm vs.24.4±5.5 mm; p=0.1) (FIG. 4 c).

HER2 Expression Status.

The in vivo immune response data for patients grouped according to levelof HER2 expression (IHC 1+, IHC 2+, or IHC 3+) were analyzed, as shownin Table 2 below. All three groups mounted substantial DTH reactionspost vaccine. Surprisingly, patients having low to intermediateexpression of HER2/neu mounted in vivo immune responses similar inmagnitude to the post-vaccine DTH responses observed in IHC 3+ patients.The low to intermediate expressing of HER2/neu patients also showed atrend toward more statistically significant differences between pre andpost-vaccine DTH responses as compared to the IHC 3+ patients.Specifically, IHC 2+ patients had a statistically significant increasein DTH response comparing pre vs. post-vaccine measurements (2.3±2 3 mmto 32.5±6.6 mm; p=0.02). Patients with IHC 1+ and IHC 3+ had a trendtoward more potent post-vaccine DTH responses, with the IHC 1+ patientscloser to statistical significance than the IHC 3+ patients (IHC1+=2.1±2.1 mm to 33.0±12.8 mm; p=0.06, and IHC 3+=3.9±3 9 mm to44.0±17.9 mm; p=0.1). When the DTH data of the low to intermediateexpressing patients were combined (“LE”) and compared to the DTH datafrom the IHC 3+ patients (“OE”), LE patients were unexpectedly observedto have a statistically significant increase in DTH response comparingpre vs. post-vaccine measurements (2.0±1.4 mm to 31.7±7.1 mm; p=0.002)as compared to the OE patients (3.9±3.9 mm to 44.0±17.9 mm; p=0.1).

TABLE 2 DTH Responses Based on HER2 Expression Level IHC 1+ IHC 2+ IHC3+ No IHC Number 7 5 5 1 GP2 pre-DTH average ± SE 2.1 ± 2.1 2.3 ± 2.33.9 ± 3.9 median (range) 0 (0-14.5) 0 (0-11.5) 0 (0-19.5) GP2 post-DTHaverage ± SE 33.0 ± 12.8 32.5 ± 6.6  44.0 ± 17.9 median (range) 23.5(0-104) 28 (22.5-58.5) 30 (14.5-114.5) p-value pre- 0.06 0.02 0.1 post(t-test) LE OE Number 13 5 GP2 pre-DTH average ± SE 2.0 ± 1.4 3.9 ± 3.9median (range) 0 (0-14.5) 0 (0-19.5) GP2 post-DTH average ± SE 31.7 ±7.1  44.0 ± 17.9 median (range) 24.0 (0-104) 30 (14.5-114.5) p-valuepre- 0.002 0.1 post (t-test)

Epitope Spreading.

Lastly, evaluation for evidence of intra-antigenic epitope spreading inresponse to vaccination with GP2+GM-CSF was performed. Measurement ofboth GP2-specific and E75-specific CTLs before, during, and aftervaccination was performed. We observed that the percentage ofE75-specific CTLs did rise significantly when we compared pre vs.maximum levels (0.8±0.2% vs. 2.0±0.2%; p=0.0001), and increased, but notsignificantly, pre vs. post-vaccine (0.8±0.2% vs. 1.2±0.2%; p=0.1) andpre vs. long-term (0.8±0.2% vs. 1.0±0.2%; p=0.6) in response tovaccination with GP2-peptide (FIG. 5). Of note, these levels ofE75-specific CTLs are similar in magnitude to primary vaccination withE75 with the only difference being a trend towards larger E75 maximumdimer responses compared to GP2 (2.0±0.2% vs. 1.4±0.2%; p=0.07).

The observation of more robust DTH and local reactions along withgreater CTL responses amongst the patients starting with higher GM-CSFdoses suggests that the immunoadjuvant dose plays a role in theimmunogenicity, and possibly the efficacy of HER2/neu peptide vaccines.As previously reported, larger doses of E75+GM-CSF led to more robustDTH reactions and trends toward fewer recurrences with improved survivalin the patients who did recur (Peoples G E et al., Clin Cancer Res(2008) 14(3):797-803). Another recent study with E75 has shown thatadministering E75 and GM-CSF in six monthly inoculations todisease-free, breast cancer patients in the optimal dose group (ODG) of1000 mcg E75 and 250 mcg GM-CSF (1000:250:6) results in an averagepost-vaccination DTH response of 21.5 mm. Holmes et al., Cancer (2008)113:1666-75. The post-vaccination DTH responses of the suboptimal dosegroup (SDG) were significantly lower than the OBD. Interestingly, thepatients in the ODG had fewer cases of disease recurrence despite havingmore aggressive disease, indicating that the DTH response provides auseful marker for clinical outcome and, in particular, for measuringpredisposition to disease recurrence, with a lower DTH correlating witha higher predisposition to disease recurrence or a shorter disease-freesurvival time and vice versa.

Surprisingly, even though GP2 has a relatively poor binding affinity forHLA-A2 and is the subdominant epitope, patients treated with GP2 andGM-CSF exhibited markedly larger DTH responses as compared to thoseinduced with the immunodominant epitope, E75 (plus GM-CSF). In thistrial with GP2, larger DTH responses were seen in patients withoutpre-existing immunity (43.9 mm) as well as patients receiving the higherGM-CSF dose (45.7 mm) Specifically, the average post-vaccination DTHresponse for all GP2+GM-CSF patients was 35.1 mm, whereas the averagepost-vaccination DTH response for E75+GM-CSF patients was 11.3 mm (SDG)and 21.5 mm (ODG). Patients treated with GP2 and 250 mcg GM-CSF had anaverage post-vaccination DTH over twice the size of the similarlytreated E75 OBD (1000:250:6) patients (45.7 mm vs. 21.5 mm)Surprisingly, in comparison to previous trials with the immunodominantpeptide E75, the average DTH reaction to GP2 was approximately twice thesize of that induced by E75 with on average half the peptide dose. Notonly do these findings further illustrate the immunogenicity of GP2 andunderscore its clinical relevance, but the in vivo DTH data alsostrongly suggest that GP2, despite being the subdominant epitope, shouldbe more effective at reducing breast cancer recurrence than E75.

Example 2 Phase II Trial of GP2+GM-CSF

Methods

Disease-free, high risk breast cancer patients who have completedstandard adjuvant therapy were enrolled at multiple sites and randomizedto receive six monthly inoculations of either 500 mcg of GP2 with 125mcg of GM-CSF (Peptide group; PG) or 125 mcg of GM-CSF alone (adjuvantgroup; AG). Toxicity was assessed after each inoculation. Immunologicresponse was monitored by measured delayed type hypersensitivityreactions (DTH) and an HLA-A2:Immunoglobulin dimer assay to detectGP2-specific CD8⁺ T-lymphocytes. Patients were monitored clinically,radiographically, and pathologically for recurrence.

Results

Thus far, 50 (27 PG, 23 AG) of the planned 200 patients have completedthe primary series. The PG and AG have similar demographic/prognosticcharacteristics (Table 3).

TABLE 3 Patients demographics and prognostic characteristics for PhaseII Study Demographics Peptide Adjuvant p= N= 27 23 Age (median) 52 510.88 Node Positive 51.9% 69.6% 0.32 Grade 3 51.9% 56.5% 1 Tumor >= 2 cm66.7% 52.2% 0.45 ER/PR neg 40.7% 43.5% 0.92 HER2 overexpress 59.3% 47.8%0.6

Toxicity profiles in the PG and AG were nearly identical with no grade4-5 local toxicities and no grade 3-5 systemic toxicities in either arm.Median DTH reaction to GP2 increased significantly from pre-vaccinationlevel after completion of the primary series (post-vaccination) in thePG group (1.0±0.8 cm to 18.0±3.1 cm; p<0.0001) and to a lesser extent inthe AG group (0.0±1.0 cm to 0.5±3.3 cm; p<0.01) (FIG. 6). Thepost-vaccination DTH was significantly larger in the PG compared to theAG (18.0±3.1 cm vs 0.5±3.3 cm, p=0.002) (FIG. 6). All (27/27) PGpatients displayed significant immunity (SI) by DTH (reaction largerthan 1 cm) post-vaccination compared to 45.5% (10/22) of AG patients. Ofthe 10 AG patients with post-vaccination SI, 50% (5/10) hadpre-vaccination SI compared to just 16.6% (2/12) without SIpost-vaccination (p=0.38). The % GP2-specific CD8⁺ lymphocytessignificantly increased from baseline at 6 months after completion ofthe primary series in the PG (0.65±0.15 to 1.82±0.23, p=0.002) and didnot change significantly in the AG (1.08±0.16 to 1.41±0.49, p=0.45).

Because this is an ongoing multi-site study, with patients enrolled on arolling basis, recurrence data are not yet complete. However, thepreliminary data show that the PG patients have experienced anapproximately 50% reduction in recurrence rate as compared to thecontrol AG patients, similar to recurrence rates observed at 24 monthsin patients treated with E75+GM-CSF (Peoples G E et al., Clin Cancer Res(2008) 14(3):797-803). More specifically, at a median follow up of 17.9months, the recurrence rate in the PG is 7.4% (2/27) compared to 13%(3/23) in the AG (p=0.65). More recurrence rate data will becomeavailable as more patients undergo follow up at 24 months and beyond andas more patients enroll in the study.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

1. A method of preventing breast cancer recurrence in a subject,comprising administering to the subject a composition in an amounteffective to prevent breast cancer recurrence, wherein the compositioncomprises a pharmaceutically effective carrier, a peptide having theamino acid sequence SEQ ID NO:2, and granulocyte macrophage-colonystimulating factor, and wherein, other than the peptide having the aminoacid sequence of SEQ ID NO:2, the composition does not contain any otherHer2/neu-derived peptides; and wherein the subject is in remissionfollowing treatment with a standard course of therapy.
 2. The method ofclaim 1 wherein the composition is administered by injection orinoculation.
 3. The method of claim 2, wherein the injection is anintradermal injection.
 4. The method of claim 2, wherein the compositionis injected in one or more split doses.
 5. The method of claim 4,wherein the injection sites on the subject are located about 5 cm apartfrom each other.
 6. The method of claim 1, wherein the composition isadministered every month for six months.
 7. The method of claim 1,further comprising administering to the subject a booster comprising aneffective amount of a vaccine booster composition comprising apharmaceutically effective carrier and a peptide having the amino acidsequence of SEQ ID NO:2.
 8. The method of claim 7, wherein the boosteris administered every six or 12 months after a primary immunizationschedule is completed.
 9. The method of claim 1, wherein the subject isa human.
 10. The method of claim 9, wherein the human expresses humanleukocyte antigen A2.
 11. The method of claim 9, wherein cancer cellsfrom the human express detectable levels of HER2/neu.
 12. (canceled) 13.The method of claim 1, wherein the granulocyte macrophage-colonystimulating factor is recombinant human granulocyte macrophage-colonystimulating factor.
 14. The method of claim 8, wherein the vaccinebooster composition further comprises an adjuvant.
 15. The method ofclaim 14, wherein the adjuvant is granulocyte macrophage-colonystimulating factor.
 16. The method of claim 1, wherein administering thecomposition induces a cytotoxic T-lymphocyte response to the peptidehaving the amino acid sequence SEQ ID NO:2.
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. The method of claim 1, wherein the subjectdoes not have pre-existing immunity to the peptide having the amino acidsequence of SEQ ID NO:2.